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. Author manuscript; available in PMC: 2023 Nov 22.
Published in final edited form as: Cell. 2023 Oct 3;186(21):4567–4582.e20. doi: 10.1016/j.cell.2023.08.041

Figure 5. Preclinical CAR T cell production via homology-directed repair results in chromosome loss.

Figure 5.

(A) Strategy to generate CAR T cells via HDR with Cas9. AAV6 encoding a 1928ζ CAR between left and right homology arms (LHA and RHA, respectively) serves as a template for HDR after Cas9 cleavage (yellow lightning bolt) of TRAC.

(B) Three potential genomic outcomes after Cas9 HDR: indels that disrupt TCR expression (top), insertion of the CAR transgene that simultaneously disrupts TCR expression (middle), and chromosome loss that disrupts TCR expression (bottom).

(C) Quantification of chromosome 14 loss enrichment across two TRAC-targeting gRNAs with or without an AAV HDR template from scRNA-seq (n = 2 biological donors). Two separate batches of CAR T cells were manufactured, before being subjected to scRNA-seq 4 or 7 days after generation. Chromosome 14 loss enrichment was calculated relative to T cells treated with Cas9 and a non-targeting gRNA.

(D) Chromosome 14 loss enrichment over time, normalized to Cas9 editing efficacy (n = 2 biological donors). Editing efficacy was determined by the percentage of TCR negative cells as measured via flow cytometry (see Figure S5E).

See also Figures S5 and S7.